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Regular Articles |
From the Molecular and Cellular Biology Research
Program,*
Sunnybrook and Women’s College Health Sciences
Centre, Toronto, Ontario, Canada; the Department of Medical
Biophysics,
University of Toronto, Toronto,
Ontario, Canada; the Hamilton Civic Hospitals Research
Centre,
McMaster University, Hamilton,
Ontario, Canada; the Samuel Lunenfeld Research
Institute,¶
Mt. Sinai Hospital, Toronto, Ontario,
Canada; the Department of Biomedical Sciences,||
Ontario
Veterinary College, University of Guelph, Guelph, Ontario, Canada; and
The Centre for Transgene Technology and Gene
Therapy,§
Flanders Interuniversity Institute
for Biotechnology, Leuven, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(HIF-1), a transcription
factor that has been shown to mediate hypoxia-induced
responses, including apoptosis. Despite reduced vascularization
in HIF-1-/- embryonic stem cell-derived
tumors, their growth in vivo was found to be
accelerated relative to HIF-1+/+ tumor counterparts. We
hypothesized that this paradoxical observation is because of decreased
apoptotic rate, resulting in diminished vascular dependence of
HIF-1-/- cells. Analysis of heterogeneous tumors
established from mixtures of HIF-1+/+ with
HIF-1-/- cells revealed that the proportion of cells
expressing wild-type HIF-1 was increased in perivascular areas and
decreased in distal tumor regions. Thus, cells expressing
HIF-1 were found to be highly dependent on proximity to blood
vessels for their growth and survival in vivo,
whereas cells that had lost HIF-1 expression were much less so.
Heterogeneity in angiogenesis dependence was also observed among cell
subpopulations isolated from human melanoma xenografts. This potential
for selection of less vascular-dependent tumor cell variants throughout
the course of disease progression may have important implications for
the long-term efficacy of anti-angiogenic therapy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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; such
treatment can result in very gradual but eventually complete regression
of such tumors, without any evidence of relapse. Similarly, chronic,
intermittent treatment of various transplantable mouse tumors with
endostatin is not compromised by manifestation of acquired resistance
to this direct-acting anti-angiogenic drug.3 There are reasons, however, to anticipate that certain types of anti-angiogenic therapy might be associated with some degree of acquired resistance.6 For example, given the redundancy of pro-angiogenic growth factors produced by tumor cells, targeting only one such growth factor should, in theory, lead to variants that can sustain an angiogenic response by producing alternative angiogenesis stimulators. This may explain why long-term treatment of transplanted tumors in mice with antibodies to the flk-1 vascular endothelial growth factor (VEGF) (type 2) receptor can lead to eventual relapse after a period of dormant growth.7 It is possible that variants having low-grade resistance to direct-acting angiogenesis inhibitors may emerge with chronic treatment as well. Thus, continuous treatment using low doses of a chemotherapeutic drug used as an anti-angiogenic agent, eg, cylophosphamide8 or vinblastine7 can result in eventual tumor relapse. Such relapses can be indefinitely delayed by the use of more potent combination anti-angiogenic therapy using an additional anti-angiogenic drug such as TNP-4708 or monoclonal flk-1 antibodies along with the chemotherapeutic drug.7 Resistance to the single treatment may be because of epigenetic changes induced in the endothelial cells or, conceivably, to the selection of tumor cell variants that have a reduced vascular dependence for survival. The latter may occur as a consequence of tumor cell heterogeneity, the subject of this study.
It is well known that cells varying in such properties as growth, invasiveness, metastatic potential, and drug resistance can be isolated from single tumors.9-14 In theory, it is clearly possible that this tumor cell heterogeneity may apply to angiogenesis, ie, variant populations may also differ in their expression of angiogenic factors, and consequently in their ability to induce angiogenesis. If this were the case, variability in blood vessel density within different regions of a tumor might be expected.15 Indeed, such areas of high vessel density, (so called "vascular hot spots") are characteristic of most tumors.16,17 Although it is clear that acquisition of the angiogenic phenotype is an important step in tumor progression,18 what has not generally been considered is the possibility that the relative dependence of tumor cells on angiogenesis itself may also become subject to the same types of selective pressures. Throughout time, the outgrowth of subpopulations of less vascular-dependent or angiogenesis-dependent malignant cells could potentially occur, particularly in the context of certain types of long-term anti-angiogenic therapy,6 with selection of such traits as an increased capacity to survive in nutrient- or oxygen-deprived areas of a tumor. Indeed, there is evidence that hypoxia provides a selection mechanism for cells with diminished susceptibility to apoptosis, as it has been shown that small numbers of cells harboring mutations in the p53 tumor suppressor gene overtake wild-type cells under hypoxic growth conditions.19
An important component of the hypoxic response is the activation of
gene expression mediated by hypoxia-inducible factor (HIF-1).
Inactivation of the HIF-1 gene in embryonic stem cells by Carmeliet
and colleagues20
has revealed a new role for this
transcription factor in the hypoxic regulation of cell growth and
apoptosis. Although apoptosis was increased in
HIF-1+/+ cells on exposure to hypoxia,
HIF-1-/- cells were found to be relatively
hypoxia-resistant. Striking differences were found in tumors derived on
injection of these ES cells into mice. Compared to
HIF-1+/+ tumors,
HIF-1-/- tumors were poorly vascularized,
deficient in large vessels, and contained more hypoxic areas. Despite
these angiogenic defects, the growth of
HIF-1-/- tumors was actually accelerated
relative to the HIF-1+/+ tumors, because of a
decrease in hypoxia-induced tumor cell apoptosis. One possible
explanation for this paradoxical coupling of increased tumor growth
rate with reduced vascularization in
HIF-1-/- tumors might be that the loss of
HIF-1 in these cells renders them less vascular-dependent.
To investigate whether HIF-1 loss might decrease vascular dependence
in these cells, and by extension allow us to study the possibility of
variable vascular dependence of tumor cell subpopulations in a more
general way, we established tumors from mixtures of
HIF-1+/+ and
HIF-1-/- cells, and examined the
distribution of each population relative to perfused blood vessels. Our
results demonstrated that HIF-1+/+ cells
predominantly and preferentially localized to areas immediately
surrounding perfused blood vessels whereas
HIF-1-/- cells were located more distal to
such vessels. We also studied heterogeneity in vascular dependence of
human tumors by selecting human melanoma variants from xenografts based
on their relative proximity to perfused vessels. Our results suggest
that cells within a single tumor may be heterogeneous with respect to
angiogenesis or blood vessel dependence, and these differences may
arise from genetic changes occurring during malignant tumor
progression.
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Materials and Methods |
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The HIF-1-/- embryonic stem (ES) cells
generated by homologous recombination, and
HIF-1+/+ ES cells harboring a randomly
integrated targeting vector have been previously
characterized.20
The yellow fluorescent protein (YFP) and
lacZ-tagged HIF-1 +/+ ES cells (YC5
and C16, respectively) were derived by one of us. The C16 cells are
functionally normal, whereas harboring a heterozygous deletion of the
FGFR2 gene. All ES cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 15% fetal bovine
serum (HyClone, Logan, UT), 0.1 mmol/L nonessential amino acids,
1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine,
50 µg/ml each penicillin and streptomycin (all Life Technologies,
Inc., Rockville, MD), 100 µmol/L ß-mercaptoethanol (Sigma Chemical
Co., St. Louis, MO) and leukemia inhibitory factor. Human melanoma
cells were grown in RPMI 1640 medium (Life Technologies, Inc.) with 5%
fetal bovine serum, and bovine aortic endothelial cells were cultured
in Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.) with
5% fetal bovine serum.
Establishment of Heterogeneous HIF-1-/- and
HIF-1+/+ Tumors
ES cell-derived tumors (teratomas) were produced by subcutaneous
injection of 5 x 106
ES cells into athymic
nude (nu/nu) mice (Charles River, Wilmington, MA). For heterogeneous
tumors, equal numbers of HIF-1-/- and
HIF-1+/+ (untagged, YFP-tagged, or
lacZ-tagged) ES cells, totaling 5 x
106
cells, were mixed before injection. Growth of
tumors was monitored until an estimated volume of at least 1,000
mm3
was reached, as calculated using the standard
formula (length x width2
x 0.5).
Labeling of Tumor Cells as a Function of Distance from Vasculature
Tumor-bearing mice were injected via the tail vein with 200 µl of 10 mg/kg Hoechst 33342 dye (Sigma Chemical Co.), which was allowed to circulate and diffuse for 5 minutes before tumor removal. Under these conditions, a reproducible perivascular tumor cell-labeling gradient was achieved, as described elsewhere.21,22 Tumor tissue was disaggregated with an enzyme cocktail containing collagenase type III (Worthington, Lakewood, NJ), hyaluronidase (Sigma Chemical Co.), and collagenase type IV (Sigma Chemical Co.), washed several times, and resuspended in phosphate-buffered saline (PBS) to produce a single cell suspension suitable for fluorescence-activated cell sorting (FACS) analysis.
Analysis of HIF-1 Genotype in Proximal and Distal Tumor Cell
Subpopulations
For HIF-1-/- and untagged
HIF-1+/+ mixed tumors, the tumor cell
suspension was sorted based on Hoechst 33342 fluorescence intensity on
an EPICS Elite V flow cytometer (Coulter Electronics, Hialeah, FL).
Gates were set to collect the 5% most brightly stained cells,
designated "proximal," and the 5% least brightly stained cells,
designated "distal," based on their relative proximity to tumor
blood vessels. Genomic DNA was isolated from the two cell
subpopulations, and the composition of each, with respect
to HIF-1 genotype, was determined by polymerase chain reaction using
EcoRI-digested DNA and primers flanking the deletion site.
The following primers were used: HIF700,
5'-CAAGCATTCTTAAATGTGGAGC-TATCT-3'; HIF960,
5'-TTGTGTTGGGGCAGTACTGGAAAGATG-3'; NEO187,
5'-CGAAGGGGCCACCAAAGAACGGAGCCG-3'. Amplification of the wild-type
allele using HIF960 and HIF700 yielded a product of 270 bp, whereas a
340-bp product was obtained by amplification of the
HIF-1-/- allele with primers HIF960 and
NEO187. Primers flanking the neo sequence were used as an
internal control, producing a product of 426 bp, and the sequences were
as follows: H1, 5'-TCCACCATGATATTCGGCAA-3'; H4,
5'-TGAATGAACTGCAGGACGAG-3'.
For mixed tumors composed of HIF-1-/- and
YFP-labeled HIF-1+/+ ES cells, the HIF-1
genotype was determined directly by flow cytometric analysis of the
tumor cell suspension. Using a FACStar Plus flow cytometer (Becton
Dickinson, Mountain View, CA) equipped with a dual laser, cells could
be analyzed simultaneously for Hoechst and YFP fluorescence intensity.
The percentage of YFP-positive HIF-1+/+ cells
among the 5% most brightly (proximal) and 5% most dimly (distal)
Hoechst-stained cells could be ascertained from the data.
In Vivo Selection of Tumor Cell Subpopulations for Varying Vascular Dependence
Tumors were established from the WM239A human melanoma cell line by intradermal injection of 106 cells into athymic nude mice. After tumor growth, mice were injected with Hoechst 33342 dye, as before, followed by euthanasia and dissection of the tumor under sterile conditions. Pieces of tumor were saved for histological assessment, and the remaining tissue digested as described. The cell suspension was sorted on an EPICS V flow cytometer (Coulter Electronics), equipped with a UV laser. Windows were set to collect the 15% most and least brightly stained cells (proximal and distal cells, respectively). Sorted cells were plated and grown in vitro, then re-injected into mice. This sequence of tumor growth, cell sorting, and establishment of sorted cells in culture constituted one round of in vivo vascular selection. The procedure was repeated for a total of four rounds to evolve the proximal and distal cell lines, respectively, selected to be close to, or distant from perfused tumor vasculature.
Histology and Immunostaining
For lacZ staining of ES cell-derived tumor tissues, specimens were fixed in lacZ fixative (0.2% glutaraldehyde, 50 mmol/L EGTA, pH 7.3, 100 mmol/L MgCl2 in 0.1 mol/L sodium phosphate, pH 7.3) for 4 hours on ice with shaking. Samples were washed in PBS, and cryoprotected in 15% sucrose in PBS followed by 30% sucrose in PBS at 4°C. Tissue was placed in Tissue-Tek OCT (Sakura) at 4°C for at least 1 hour before freezing over dry ice. Cryosections of 10 µm were cut and placed on silanized slides.
Slides were fixed in cold PBS containing 0.2% glutaraldehyde, then washed in lacZ wash buffer (2 mmol/L MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet-P40 in 0.1 mol/L sodium phosphate, pH 7.3). Staining was performed in lacZ stain solution (0.5 mg/ml X-gal, 5 mmol/L potassium ferrocyanide, and 5 mmol/L potassium ferricyanide in lacZ wash buffer) at 37°C for 4 hours, protected from light. To identify blood vessels, slides were washed in PBS after lacZ staining, and stained with a rat anti-CD31 antibody (Pharmingen, La Jolla, CA) used at a dilution of 1:200. Immunoreactivity was visualized by incubation with 3-amino-9-ethylcarbazole (Zymed, South San Francisco, CA).
For the vascular selection experiments, tumors produced by cells from the final round of vascular selection, and tumors produced by the original unsorted WM239A cells were characterized as follows: tumor tissue was fixed in modified Carnoy’s (60% ethanol, 30% chloroform, 10% acetic acid) or 3% paraformaldehyde. Cryostat sections (6 to 8 µm) were cut and viewed under UV epifluorescence to visualize Hoechst staining. Paraffin sections (6 µm) were stained with an antibody to Von Willebrand Factor (DAKO, Carpinteria, CA) to detect vascular endothelium. Immunocomplexes were visualized by incubation with diaminobenzidine tetrahydrochloride (Boehringer Mannheim, Indianapolis, IN) or 3-amino-9-ethylcarbazole, and slides were lightly counterstained with hematoxylin.
Analysis of WM239 Melanoma Cell Lines and Tumors
Vascular density was assessed in tumor sections immunostained for von Willebrand Factor according to established methods.23 To assess tumor cell apoptosis, we quantified the number of apoptotic bodies in 10 nonnecrotic oil immersion fields of hematoxylin and eosin stained slides. Apoptotic bodies were characterized as highly pkynotic nuclear fragments, associated with shrunken cells.
The angiogenic activity of WM239 parent, proximal-4 (P-4), and distal-4 (D-4) cells were quantified by examining the effects of medium conditioned by these cells for 24 hours, and control medium, on bovine aortic endothelial cell proliferation in vivo. Briefly, 2.5 x 104 bovine aortic endothelial cells were plated into 12-well plates in complete media, and after 16 hours replaced with test media. Cells were incubated for an additional 72 hours, followed by cell counting.
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To distinguish cells located in perivascular areas from cells
located in more hypoxic regions of a tumor, we adapted a technique
previously used to analyze drug response of cells within solid tumors
and spheroids.21,24
The nontoxic, fluorescent, DNA-binding
dye Hoechst 33342 was injected intravenously into tumor-bearing mice at
nonsaturating concentrations. By allowing the dye to circulate for a
short time before tumor removal, cells were fluorescently labeled based
on their relative distance from perfused blood vessels (Figure 1)
. Cells immediately surrounding tumor
vasculature were highly fluorescent, whereas cells located farther away
did not fluoresce, or had a much lower level of fluorescence intensity.
After disaggregation of the tumor cells into a single cell suspension,
the sample was analyzed for Hoechst fluorescence by flow cytometry and
the cells showing the 5% highest and lowest Hoechst fluorescence
intensities were designated proximal or distal relative to perfused
vasculature.
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+/+ and HIF-1-/- Cells
in Mixed Tumors
Once the proximal and distal cell populations in the tumor were
identified, the proportion of HIF-1+/+ and
HIF-1-/- cells in these populations was
determined. First, tumors generated by injecting a mixture of
YFP-labeled wild-type ES cells with untagged
HIF-1-/- cells were analyzed. By studying
the tumor cell suspensions simultaneously for Hoechst and YFP
fluorescence, the proportion of HIF-1+/+
YFP-fluorescing cells in the proximal and distal population was
determined directly. In every tumor examined, it was found that on
removal 
4 weeks after injection of cells, the overall proportion of
HIF-1+/+ cells was decreased. Although the
original mixture injected was composed of wild-type and
HIF-1-/- cells in equal numbers, the final
percentage of HIF-1+/+ cells was diminished to
an average of only 12%, and in some tumors was as low as 4 to 5%. As
expected, analysis of control tumors derived from YFP-tagged
HIF-1+/+ cells alone showed some loss of YFP
expression because of the tumor cell isolation protocol. At completion
of the experiment, the percentage of YFP-positive cells was decreased
from 100 to 76%. However, even if a similar reduction in YFP
expression in mixed tumors is taken into account, the overall decrease
in YFP-tagged HIF-1+/+ cells in mixed tumors
from 50 to 12% (more than fourfold) remains statistically significant
(t-test, P < 0.005).
There were also dramatic differences in the distribution of
HIF-1+/+ and
HIF-1-/- cells relative to blood vessels.
HIF-1+/+ cells accounted for only 4% of
distal cells, compared to 29% of the proximal cell population (Figure 2A)
. In contrast, YFP-positive cells in
control tumors were not preferentially localized to the perivascular
tumor regions: whereas the perivascular tumor cell population was
77.6% YFP-positive, the distal population was 74.8% YFP-positive, a
difference that is not statistically significant. The significant
sevenfold increase (P < 0.002) of
HIF-1+/+ cells in proximal regions of mixed
tumors indicates that HIF-1+/+ cells localize
primarily around perfused vessels, and suggests that throughout time,
these cells might be dependent on, and/or selected by blood vessel
proximity. In contrast, cells lacking HIF-1 seem to be much less
dependent on the vasculature.
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+/+ Genotype in Tumor Regions
Distant from the Vasculature
A more direct analysis of the predominant genotype within proximal
and distal cell populations was undertaken, using polymerase chain
reaction to distinguish between the wild-type and disrupted
HIF-1 gene. This analysis could be expected to be
unaffected by fluctuations in gene expression and cell viability during
tumor growth and experimental procedures. As shown in Figure 2B
,
amplification of the wild-type gene yields a product of 270 bp whereas
a larger product of 340 bp is amplified from
HIF-1-/- DNA. As expected, both bands were
detected in DNA isolated from unsorted mixed tumor cells, indicating
the presence of both HIF-1+/+ and
HIF-1-/- cells. However, on sorting of these
cells into proximal and distal populations based on Hoechst
fluorescence, it was found that whereas both bands were detected in
proximal cell DNA, the HIF-1+/+-specific band
was absent or much weaker in the distal cell DNA. These results again
demonstrate that HIF-1+/+ cells localize
predominantly to regions surrounding perfused blood vessels and thus
seem to be more vessel-dependent than
HIF-1-/- cells, which were prevalent in both
cell populations.
To directly visualize the differential perivascular distribution of
HIF-1+/+ and
HIF-1-/- cells in mixed tumors in a
Hoechst-independent manner, lacZ-tagged
HIF-1+/+ cells were used. The localization of
these wild-type cells relative to tumor blood vessels was determined by
histochemical staining for lacZ expression accompanied by
immunostaining for CD31 as a marker of the tumor vasculature. As shown
in Figure 3
, blue
lacZ-positive HIF-1+/+ cells were
observed scattered throughout the tumor section, with decreased numbers
relative to unstained HIF-1-/- cells. This
is expected, as a reduction of their overall contribution to only 12%
of tumor cells was demonstrated in earlier experiments using the
YFP-tagged HIF-1+/+ cells. Furthermore,
although present throughout the tumor, the
HIF-1+/+ cells showed a tendency to cluster
around vascular structures, directly confirming the results that were
obtained previously with the differentially Hoechst-labeled
disaggregated tumor cells.
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+/+ cells
localize to areas proximal to the vasculature strongly suggests that
these cells, which are more hypoxia-sensitive, seem to have an
increased dependence on blood vessels for survival. Conversely, cells
that have lost HIF-1, and which are known to exhibit a decreased
susceptibility to hypoxia-induced growth inhibition and
apoptosis,20
seem to be much less vascular-dependent. Vascular Selection of Human Melanoma Xenografts
To examine whether heterogeneity in vascular dependence might
exist in the more relevant context of a human tumor, a reverse
experiment was performed. An unbiased vascular selection strategy was
devised to segregate cells in human tumor xenografts based on their
proximity to perfused microvasculature. The human melanoma cell line
WM239A was chosen for this selection, and proximal and distal cell
populations were sorted from xenografted tumors based on differential
Hoechst 33342 fluorescence as described earlier. However, the sorted
cells were passaged in culture, followed by re-injection into mice to
generate tumors, whereupon tumors were once again removed and sorted.
To establish the proximal cell line, only sorted proximal cells were
retained and propagated, and to derive the distal cell line, only
distal cells were used. This sequence of tumor growth, cell sorting,
and establishment of sorted cells in culture was repeated for a total
of four rounds, to produce the final proximal and distal cell lines
(Figure 4)
. This process allowed
progressive enrichment for tumor cell subpopulations derived from
perivascular or hypoxic areas of the tumor.
|
. Distal cell populations
showed enhanced tumor growth relative to the parental WM239A cells
after two rounds of vascular selection, which did not increase with
subsequent iterations. These cells showed no differences in tumor take
compared to parental cells. In contrast, proximal cells showed greatly
reduced growth rates in vivo. By three rounds of selection,
this reduced growth was significantly different from parental cells,
and continued to decrease with subsequent selection. Tumor take also
decreased; by the fourth round of selection, only one of five mice had
tumors after 2 weeks, and two of five mice did not develop detectable
tumors even after 20 weeks. There was no difference in growth rates of
any of these cell lines under monolayer culture conditions.
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. Quantification of blood vessels unexpectedly indicated
significantly higher numbers per hot spot in tumors derived from the
less tumorigenic P-4 cells than in more aggressive tumors from D-4
cells (Table 1)
. Tumors from parental
WM239 cells had intermediate vascular densities. These differences in
blood vessel density may be reflective of either increase in angiogenic
proficiency of P-4 cells or their inability to proliferate or survive
distant from blood vessels. Our results support the latter
interpretation, as analysis of apoptosis in these tumors showed
significantly more apoptotic cells in P-4 tumor sections than in D-4
sections (P < 0.05, Table 1
). Furthermore, we
examined the effects of medium conditioned by WM239 parent, P-4, and
D-4 cells on the proliferation of bovine aortic endothelial cells
in vitro, and we observed no significant differences between
the cell lines (Table 1)
. There was also no significant difference in
VEGF protein secretion by these cells under either normoxic or hypoxic
conditions as measured by enzyme-linked immunosorbent assay (data not
shown). As P-4 cells were therefore not found to be more
angiogenic than their D-4 counterparts, we interpret the differences in
vascular density as an indirect measure of differential vascular
dependence between the tumor cell lines.
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Discussion |
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Tumor cells require blood vessels to supply them with metabolites
such as oxygen and glucose. Hypoxic regions are a common and
significant feature of tumors, because the disorganized vasculature is
often insufficient to meet the needs of a rapidly proliferating tumor
mass. Hypoxia occurs in tumor tissue that is >150 to 200 µm away
from a functional vessel.25
In some tumors, this blood
vessel dependence may be manifested in the formation of cuffs of viable
tumor cells tightly surrounding a single central blood vessel, with
areas of necrosis occurring beyond the critical oxygen diffusion
distance. However, some of the genetic and epigenetic changes in tumor
cells may result from adaptive and selective processes induced by
protracted focal ischemia. Hypoxia induces apoptosis in oncogenically
transformed cells through a p53-dependent pathway,26
and
thereby selects for tumor cells that have lost functional p53,
resulting in increased survival capacity. Furthermore, p53 influences
the hypoxic response of cells through regulation of HIF-1
stability.27
Oncogenic transformation itself may also
facilitate adaptation to hypoxia, for instance Ras-transformation and
hypoxia synergize to induce VEGF expression.28
The concept
of vascular dependence therefore potentially encompasses a broad range
of cellular functions, in particular cell proliferation and cell
survival under conditions of metabolic stress such as hypoxia and
hypoglycemia, as well as responses to endothelial cell-derived
paracrine signals.19,22
An Experimental Approach to Studying Heterogeneous Vascular Dependence of Tumor Cells and a Possible Genetic Basis of the Phenomenon
It has been previously shown that ES cells genetically disrupted
in the HIF-1 locus are relatively resistant to hypoxic stress,
indicating a new role for HIF-1 in mediating hypoxia-induced
apoptosis, beyond functioning in the homeostatic response to cellular
hypoxia.20
Paradoxically, although tumors derived from
HIF-1-/- cells were found to express low
levels of VEGF along with significantly reduced vascular density and
function, they nevertheless exhibited an accelerated rate of tumor
growth relative to wild-type HIF-1+/+ cells
because of a reduced rate of hypoxia-induced growth arrest and
apoptosis. As mentioned above, certain other naturally occurring
genetic changes during tumor progression may result in operationally
similar outcomes of decreased reliance on oxygen and growth factors,
and therefore diminished relative vascular dependence.6
To examine the possibility that hypoxia response may influence cellular
selection within heterogeneous tumor cell populations, tumors were
established from mixtures of hypoxia-sensitive
HIF-1+/+ and hypoxia-resistant
HIF-1-/- cells. Tumor cells were then
separated as a function of distance from blood vessels by intravenous
injection of Hoechst 33342 dye. This procedure yields brightly
fluorescent cells proximal to perfused vasculature, and a lower
fluorescence intensity in more distal tumor regions.21,24
These proximal and distal cells were distinguished by FACS, and
analyzed to determine the proportions of
HIF-1+/+ and
HIF-1-/- cells in each cell population.
Assuming one subpopulation of tumor cells differed from another in
terms of vascular/angiogenesis dependence, one would expect to find a
nonrandom distribution of these cells within the tumor. In particular,
the more vascular-dependent cell type would likely be located to a
greater extent in areas surrounding blood vessels than in the more
hypoxic and distal regions simply because such cells would have a
greater chance to die of hypoxia-induced apoptosis. It should be noted
that oxygenation in tumor vessels has been shown to be
heterogeneous29
and hence this argument would apply only
to areas surrounding well-perfused capillaries. This is also the most
significant limitation of the Hoechst technique, in that only vessels
that are perfused at the time of injection are accessible to the dye.
Consequently, fluctuations in vessel perfusion throughout time or space
might theoretically result in some of the proximal, vascular-dependent
cells being caught in the hypoxic zones of the tumor.
Despite these reservations, in the HIF-1 model system, the
HIF-1+/+ (hypoxia-sensitive) cells did indeed
show increased perivascular localization compared to the relatively
hypoxia-resistant HIF-1-/- cells. This was
shown by three independent methods. First, FACS analysis showed that
the proportion of YFP-tagged wild-type
(HIF-1+/+) cells was increased by sevenfold in
areas close to perfused vasculature. Analysis of proximal and distal
cell DNA by polymerase chain reaction to determine HIF-1 genotype
confirmed these results. Although HIF-1-/-
cells were found in both the proximal and distal cell populations,
HIF-1+/+ cells were detected in regions
proximal to vessels, but were scarce or even absent in the more distal
regions. Second, the nonrandom localization of wild-type cells was also
visualized directly by histochemical staining of lacZ-tagged
HIF-1+/+ cells in tumor sections, which
clearly showed clustering of these cells around the vasculature.
Finally, HIF-1-/- cells showed an overall
selective growth advantage in mixed tumors, with their proportion
increasing from 50% at the time of cell injection, to almost 90% at
the time of tumor removal, consistent with their previously described
increased malignant properties.20
An analogous trend has
also been observed in a series of Ras-transformed cell lines differing
in malignant potential.22
Similarly, a more aggressive
subline was derived by a vascular selection procedure from WM239
melanoma xenografts, which also contain a less aggressive, blood
vessel-associated (ie, dependent) cell population. All of the
approaches consistently support the notion of greater malignancy of
cells that are able to survive within hypoxic regions of a tumor. Such
cells might be considered less angiogenesis-dependent.
The results obtained with the murine ES cell-derived teratomas should
not be interpreted as a suggestion that HIF-1 loss itself may be
favorable for tumor progression. HIF-1-/-
cells merely represent a phenocopy of hypoxia-resistant tumor cells. In
other words, loss of HIF-1 may not be a favored pathway leading to
hypoxia-resistant cellular phenotype in human tumors. Indeed, there is
growing evidence that the opposite seems to be true, as a recent study
showed that HIF-1 is overexpressed in many common human cancers and
their metastases.30
Because HIF-1 seems to be important
for the vascularization of tumors, it is likely that its activity may
be maintained at a level sufficient for expression of genes such as
VEGF, but without induction of apoptosis. However, our experiments
demonstrate that in principle, cells harboring certain defined genetic
defects that promote their survival under stressful conditions such as
hypoxia, show differences in angiogenesis dependence, as indicated by
their nonrandom localization relative to tumor blood vessels. The
HIF-1 status of the sublines derived from WM239 melanoma was
investigated by Western blotting, and no difference in protein
expression was detected between P-4 and D-4 cells when cultured in the
presence of cobalt chloride to mimic hypoxia (data not shown). However,
other elements of HIF-1 signaling (DNA binding, gene transcription)
have not been investigated in detail and the role of this mechanism in
vascular selection cannot be definitively ruled out. It is also
possible that this selection may be driven by more complex sets of
mechanisms including negative pressures such as acidification, glucose,
or growth factor deprivation in conjunction with hypoxia.
Implications for Anti-Angiogenic Therapy of Tumors
Our results clearly have potentially important implications for anti-angiogenic therapy. First, it might be predicted that anti-angiogenic drugs would preferentially affect tumor cells immediately adjacent to tumor vessels rather than those located in hypoxic regions more distal to such vessels. Indeed, this is exactly what was reported by Bergers and colleagues.31 Thus, treatment of islet cell pancreatic tumors arising in transgenic oncomice with a combination of angiostatin and endostatin, or TNP-470, also resulted in tumor regressions that were accompanied by apoptosis of tumor cells found in close apposition to tumor capillaries. As noted by Bergers and colleagues,31 this observation is counterintuitive as one might have expected preferential apoptosis of the hypoxic cells after such a therapeutic intervention. Our results help explain such findings. Second, although tumors will likely never become entirely angiogenesis-independent, the degree to which they rely on blood vessels could conceivably decrease throughout time. Therefore, over the course of long-term anti-angiogenic therapy, tumors may become less responsive to treatment with certain anti-angiogenic drugs, especially when used as single agents.6 Hence, it may be necessary to increase the intensity of anti-angiogenic treatments, for example, by using combination treatment regimens. We successfully used such a strategy by combining continuous low-dose vinblastine chemotherapy (as an anti-vascular targeting strategy) with anti-flk-1 antibodies.7 This drug combination did not result in acquired drug resistance, ie, the emergence of angiogenesis-independent tumor cell variants, despite a very long term (eg, 6 months) of continuous treatment of human neuroblastoma xenografts or mouse tumors.7,8 Similarly, a combination of angiostatin and endostatin is clearly superior to either drug used alone as an anti-angiogenic treatment strategy.31,32
The success of such combinations as continuous low-dose chemotherapy and anti-flk-1 antibodies may be related to the necessity for neutralizing varioius resistance factors for activated endothelial cells of new blood vessels. Indeed, VEGF itself may be one such resistance factor,33 eg, by up-regulating anti-apoptosis genes such as bcl-2, A1, XIAP, and survivin in endothelial cells.34-37
In summary, this study suggests that tumor cell heterogeneity may apply to angiogenesis, ie, cells within human and rodent tumors may differ in their vascular dependence, and such differences could, in principle, arise from genetic alterations occurring during cancer progression, eg, changes that affect the apoptotic response of a tumor cell to hypoxia. Contrary to what is often believed, high blood vessel density may not always be indicative of high angiogenic capacity of a given tumor, but may also, at least in some cases, signify the inability of less malignant tumor cells to thrive beyond certain short distances from their capillaries. A relative loss of vascular dependency among tumor cells may therefore constitute a significant determinant in promoting malignant progression, with possible implications for metastatic potential, as well as having important consequences for the long-term efficacy of certain types of anti-angiogenic therapy.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Medical Research Council of Canada (Canadian Institutes of Health Research), the National Institutes of Health, United States (CA-41233 to R. S. K.), and the National Cancer Institute of Canada (11162 to B. L. C.). J. L. Y. is the recipient of a Medical Research Council of Canada doctoral research award.
Accepted for publication January 5, 2001.
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